The present invention is generally in the field of polyhydroxyalkanoate materials, and more particularly to improved methods of production thereof.
Polyhydroxyalkanoates (PHAs) are natural, thermoplastic polyesters and can be processed by traditional polymer techniques for use in an enormous variety of applications, including consumer packaging, disposable diaper linings and garbage bags, food and medical products. Methods which can be used for producing PHA polymers from microorganisms which naturally produce polyhydroxyalkanoates are described in U.S. Pat. No. 4,910,145 to Holmes, et al.; Byrom, “Miscellaneous Biomaterials” in Biomaterials (Byrom, ed.) pp. 333-59 (MacMillan Publishers, London 1991); Hocking and Marchessault, “Biopolyesters” in Chemistry and Technology of Biodegradable Polymers (Griffin, ed.) pp. 48-96 (Chapman & Hall, London 1994); Holmes, “Biologically Produced (R)-3-hydroxyalkanoate Polymers and Copolymers” in Developments in Crystalline Polymers (Bassett, ed.) vol. 2, pp. 1-65 (Elsevier, London 1988); Lafferty et al., “Microbial Production of Poly-b-hydroxybutyric acid” in Biotechnology (Rehm & Reed, eds.) vol. 66, pp. 135-76 (Verlagsgesellschaft, Weinheim 1988); Müller & Seebach, Angew. Chem. Int. Ed. Engl. 32:477-502 (1993). The natural biosynthetic pathway for production of polyhydroxybutyrate (PHB) is shown in FIG. 1.
Methods for producing PHAs in natural or genetically engineered organisms are described by Steinbüchel, “Polyhydroxyalkanoic Acids” in Biomaterials (Byrom, ed.) pp. 123-213 (MacMillan Publishers, London 1991); Williams & Peoples, CHEMTECH, 26:38-44 (1996); Steinbüchel & Wiese, Appl. Microbiol. Biotechnol., 37:691-97 (1992); U.S. Pat. Nos. 5,245,023; 5,250,430; 5,480,794; 5,512,669; 5,534,432 to Peoples and Sinskey (which also disclose and claim the genes encoding reductase, thiolase, and PHB polymerase); Agostini et at, Polym. Sci., Part A-1, 9:2775-87 (1971); Gross et al., Macromolecules, 21:2657-68 (1988); Dubois, et al., Macromolecules, 26:4407-12 (1993); Le Borgne & Spassky, Polymer, 30:2312-19 (1989); Tanahashi & Doi, Macromolecules, 24:5732-33 (1991); Hori et al., Macromolecules, 26:4388-90 (1993); Kemnitzer et al., Macromolecules, 26:1221-29 (1993); Hori et al., Macromolecules, 26:5533-34 (1993); Hocking & Marchessault, Polym. Bull., 30:163-70 (1993); Xie et al., Macromolecules, 30:6997-98 (1997); and U.S. Pat. No. 5,563,239 to Hubbs et al. A general pathway for production of PHAs is shown in FIG. 2. Synthetic polymer synthesis approaches including direct condensation and ring-opening polymerization of the corresponding lactones are described in Jesudason & Marchessault, Macromolecules 27:2595-602 (1994); U.S. Pat. No. 5,286,842 to Kimura; U.S. Pat. No. 5,563,239 to Hubbs et al.; U.S. Pat. No. 5,516,883 to Hori et al.; U.S. Pat. No. 5,461,139 to Gonda et al.; and Canadian Patent Application No. 2,006,508. WO 95/15260 describes the manufacture of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) films, and U.S. Pat. Nos. 4,826,493 and 4,880,592 to Martini et al. describe the manufacture of PHB and PHBV films. U.S. Pat. No. 5,292,860 to Shiotani et al. describes the manufacture of the PHA copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHH).
To date, PHAs have seen limited commercial availability, with only the copolymer PHBV being available in development quantities. This copolymer has been produced by fermentation of the bacterium Ralstonia eutropha. Fermentation processes for production of other PHAs have been developed (Williams & Peoples, CHEMTECH 26: 38-44 (1996)). Plant crops are also being genetically engineered to produce these polymers, offering a cost structure in line with the vegetable oils and direct price competitiveness with petroleum-based polymers (Williams & Peoples, CHEMTECH 26: 38-44 (1996)).
Several factors are critical for economic biological production of PHAs, including substrate costs, fermentation time, and efficiency of downstream processing. For large-scale fermentations of commodity products, it is generally known that plasmid-based systems are unsatisfactory due to the extra burden of maintaining the plasmids and problems in maintaining stable expression.
Known biological systems for the production of PHAs containing 3-hydroxy-co-hydroxyhexanoate (3H-co-HH) are inefficient. For example, Shimamura, et al., Macromolecules, 27:878 (1994) discloses that Aeromonas caviae synthesizes a PHA composed of 3-hydroxybutyrate and 3-hydroxyhexanoate (3HH) when grown on olive oil or C12 to C18 fatty acids. The fraction of the 3HH monomer was determined to be dependent on the concentration of the carbon source and the fermentation time and could amount to levels of 25% (Doi, et al., Macromolecules, 28: 4822 (1995)). As a result of increasing 3HH substrate levels, the crystallinity, melting temperature, and glass-transition temperature of the PHA decrease. These changes in physical properties lead to an increased susceptibility to degradation by PHB depolymerase from Alcaligenes faecalis. Other natural microorganism that incorporate low levels of 3HH in a PHB copolymer are Comamonas testosteroni and Bacillus cereus (Huisman, et al., Appl. Environ. Microbial. 55: 1949 (1989); Caballero, et al., Int. J. Biol. Macromol., 17: 86 (1995)). Recombinant Pseudomonas putida GPp104 strains in which the phb genes from either Thiocapsia pfenigii or Chromatium vinosum were introduced also accumulated PHA with 3-hydroxyhexanoate as major constituent.
PHAs generally are divided into two classes based on the polymer composition: short side-chain PHAs and long side-chain PHAs. Incorporation of monomers from one group into a PHA belonging to the other usually is limited to low levels. In some cases where the monomers are abundant for both PHAs, the bacterium generally produces separate PHA granules each comprising one type of PHA. Substrate specificities of the PHA polymerases therefore can be generalized as optimal for short side-chains (C4 and C5) or medium side-chains (C8-C10). Based on composition of PHAs synthesized by individual microorganisms, PHA polymerases that incorporate 3-hydroxyhexanoate can be identified. Thus, PHA polymerases from A. caviae, C. testosteroni and T. pfenigii are known for incorporating 3-hydroxyhexanoate into the PHA, whereas the enzymes from Paracoccus denitrificans, Sphaerotilus natans and Rhodococcus sp. have a preference for 3-hydroxyvalerate. The PHA polymerases from the latter organisms also are useful in making PHB-co-HH copolymers, due to their preference for C5 over C4. Unfortunately, however, these bacteria generally have a low growth rate, often are difficult to break open, and have only a limited amenability to genetic engineering. It is thus desirable to develop efficient, more cost-effective ways of producing PHAs containing 3H-co-HH by biological systems.
It is therefore an object of the present invention to provide genetically engineered systems for the production of polyhydroxyalkanoate polymers including 3-hydroxyhexanoate monomers (HHPHA).
It is another object of this invention to provide useful mutations which can be used to produce 3-hydroxyhexanoic monomers from more economic feedstocks, such as butyrate or butanol.
It is a further object of this invention to provide genes suitable for converting cellular metabolites derived from carbohydrate feedstocks to Butyryl-CoA for the production of 3-hydroxyhexanoate comonomers.
It is another object of this invention to provide improved methods of producing PHAs containing 3-hydroxyhexanoate as comonomer.
It is still another object of this invention to provide new pathways in biological systems for the endogenous synthesis of the 3-hydroxyhexanoate monomer.
It is a further object of this invention to provide genetically engineered biological systems for production of PHAs containing 3-hydroxyhexanoate in which expression is sufficient and stable.